AU2001243964A1 - Composition and method for the prevention and/or the treatment of allergy - Google Patents

Description

COMPOSITION AND METHOD FOR THE PREVENTION AND/OR THE

TREATMENT OF ALLERGY

Field of the invention

[0001] The present invention is related to a composition and a method for modulating the immune response of a patient to an allergen and consequently preventing and/or treating allergy, especially airborne and foodborne allergies, as well as diseases of allergic origins.

State of the art

[0002] Allergic diseases have a major impact on public health, with more than 20% of the general population affected by at least one form of allergic manifestations, such as rhinitis, bronchial asthma, atopic eczema or systemic anaphylaxis to drugs or insect stings. [0003] Allergy, which is also called immediate hypersensitivity, s mediated by specific antibodies belonging to the IgE isotype. The mechanisms by which an allergic reaction develops are well elucidated, but the factors predisposing to allergy are poorly understood, as well as the factors governing the evolution during aging. [0004] The manifestations of allergy often evolve from atopic eczema m infancy to airway hypersensitivity and bronchial asthma m later infancy and childhood. Atopic eczema is thought to be at least partly related to the development of allergy to food antigens, while allergic asthma is by definition triggered by sensitization to airborne allergens.

[0005] Whether sensitization to food- and airborne allergens represent two independent manifestations of an allergic status, namely a predisposition to produce IgE antibodies to common antigens, or whether the two phenomena are linked together is unknown.

[0006] Current treatment of allergic symptoms include allergen avoidance, drug therapy and immunotherapy . Complete avoidance from allergen exposure is the most logical approach, but it remains very difficult, or even impossible, to achieve m most cases. Drug therapy is useful, but alleviates symptoms without influencing their causes. In addition, drug treatment is usually limited by undesirable side-effects.

[0007] There are currently different approaches for immunotherapy. Among these approaches is conventional hyposensitisation which is a treatment consisting m administering to the patient progressively increasing doses of the allergen (s) to which said patient has developed a sensitivity.

[0008] Another approach consists m altering the physico-chemical properties of the allergen in order to reduce recognition by specific antibodies, IgE m particular.

[0009] It is also possible to use allergen-derived peptides. Such peptides are presented by antigen-presenting cells to specific T cells m a manner which results m induction of T cell unresponsiveness , or anergy. Under such circumstances no specific IgE antibodies are produced. Patent application WO 93/08279 gives an example of such allergen-derived peptides. One human application of this concept is the administration of a peptide derived from the sequence of T cell epitopes present on the Fel dl allergen, by subcutaneous injections m cat-sensitive individuals

(Wallner B.P & Gefter ML (1994), Allergy 49, 302-308).

[0010] It is also possible to prevent the release of the mediators responsible for allergic manifestations by interfering m the activation signal provided to mast cells or basophils by cross-linking surface IgE antibodies. This can be achieved by using molecules presenting only one IgE binding epitope, or peptides that interfere m the recognition of IgE antibodies by the alpha chain of the high-affmity receptor.

[0011] Other methods for treating and/or preventing hypersensitivity to allergens are disclosed, for example, m documents WO 00/50044 and US 6,090,386.

Aims of the invention

[0012] The present invention aims to provide a new composition and method for modulating and modifying the immune response of a patient to an allergen and consequently providing alternative methods and means for preventing and/or treating allergy, especially airborne and foodborne allergies, as well as diseases of allergic origins .

[0013] More particularly, the present invention aims to provide such method and means which are safe and can be easily monitored and controlled.

Definitions

[0014] The following definitions are given m order to make clear the description of the present invention. [0015] It is meant by "antigen" a structure of a macromolecule, preferably made of proteic composition but also made of one or more hapten(s) , and comprising "epitopes" . [0016] It is meant by "food or pharmaceutical antigen" , an antigemc structure preferably of proteinic and/or polysacchaπdic composition, naturally available m a food component or a pharmaceutical product including a vaccine.

[0017] It is meant by "epitope" , one or several portions (which may define a conformational epitope) of an antigen or an immunogen which are specifically recognized and bound by an antibody or a portion thereof (Fab', Fab2 ' , etc.) or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able by said binding to induce an immune response.

[0018] More particularly, it is meant by " T cell epitope" an epitope which is specifically recognized and bound by a receptor at the cell surface of a T lymphocyte. [0019] It is meant by "immunogen" a food or pharmaceutical antigen which is able as a whole to trigger both a humoral and a cellular immune response. [0020] It is meant by "atopy", a predisposition (partly of genetic origin) of an individual patient having an immune system producing an excess of antibodies belonging to the IgE isotype m response to allergens. Individuals presenting such characteristics are therefore called "atopies" . [0021] An "allergen" is defined as a substance, usually a macromolecule of proteic composition, which elicits the production of IgE antibodies m predisposed, preferably genetically disposed, individuals (atopies) patients . [0022] Similar definitions are presented m the following references : Clm . Exp . All ergy, No. 26, pp. 494- 516 (1996); Mol . Biol . of Allergy and Immunology, ed . R . Bush, Immunology and Allergy Clini cs of North American Series (August 1996) . [0023] These allergens are preferably the main allergens which are selected from the group consisting of :

- food allergens present m peanuts, codfish, egg white, soybean, shrimp, milk and wheat, - house dust mites allergens obtained from Dermatophagoides spp . pteronyssmus , farmae and microceras, Euroglyphus maynei or Blomia,

- allergens from insects present m cockroach or hymenoptera, - allergens from pollen, especially pollens of tree, grass and weed,

- allergens present m animals, especially m cat, dog, horse and rodent,

- allergens present m fungus, especially from Aspergillus, Alternaπa or Cladospoπum, and

- occupational allergens present m such products as latex, amylase, etc.

[0024] Said allergens can also be mam allergens present m moulds or various drugs such as hormones, antibiotics, enzymes, etc. (See also the definition m Clm. Exp. Allergy no.26, p:494-516 (1996) and Mol . Biology of Allergy and Immunology, Ed. R. Bush (August 1996)) . [0025] "Allergy" is the ensemble of signs and symptoms observed whenever an atopic individual patient encounters an allergen to which he has been sensitised, which may result m the development of various diseases, m particular respiratory diseases and symptoms such as bronchial asthma.

[0026] "Hypersensitivity" is an untoward reaction produced m an individual upon exposure to an antigen to which it has become sensitised; "immediate hypersensitivity" depends of the production of IgE antibodies and is therefore equivalent to allergy [0027] The wording "proteins phylogenetically unrelated" refers to proteins for which no evolutionary link can be established.

Summary of the invention

[0028] The present invention is related to a method for modulating (modifying the reactivity of) the immune system of a mammal patient (including a human) towards an allergen, said patient being preferably an atopic patient [0029] Said method comprises the step of inducing a pre-sensitization of the immune system (through contact with immune cells (APC) of said patient) towards an immunogen derived from a naturally occurring antigen (which means an immunogen present m a food or pharmaceutical component) , m order to modify the immune response to a further sensitization to said allergen, such that the immune response towards the allergen is modulated

(preferably decreased m order to change the atopy behaviour of the patient) , no allergy towards said allergen develops m said patient or said allergy decreases and said patient is rendered non-allergic.

[0030] The inventors have observed that it is possible to modify the reactivity of the immune system towards said allergen (render said patient non-allergic) , because they have identified that an immunogen deriving

(obtained) from a naturally occurring antigen (presented m a food or pharmaceutical product) , could carry a T cell epitope homologous and functionally similar to an epitope present on said allergen. [0031] It is meant by an homologous T cell epitope presented m an immunogen, a T cell epitope having an ammo-acid sequence which presents more than 40%, preferably more than 50%, more preferably more than 60% homology (or sequence identity) with the corresponding epitope sequence presented an allergen.

[0032] The T cell epitope present on said allergen and the T cell epitope present on the immunogen are recognised by the same T cells, m particular by T cell clones. They could therefore be used alternatively for modulating the immune response by one compound against another .

[0033] The above-mentioned T cell epitope carried by the immunogen is preferably a universal T cell epitope, preferably present upon an airborne allergen or a foodborne allergen .

[0034] Preferably said allergen is selected from the group consisting of rhmo-smusitis allergens, allergic bronchial asthma allergens and atopic dermatitis allergens.

[0035] Preferably said allergen is the Der p II protein and the corresponding universal T cell epitope corresponds to SEQ ID NO. 1.

[0036] According to the invention, the lmmunogens are preferably derived (obtained) from myoglobm protein and the corresponding identified epitope of myoglobm is preferably SEQ ID NO. 2.

[0037] The method according to the invention comprises also the step of administrating, preferably to said mammal patient including humans, a sufficient amount of a pharmaceutical composition comprising said immunogens or the above-mentioned epitopes of said immunogens and a pharmaceutical adequate carrier suitable for an administration to said patient. The pharmaceutical adequate carrier can vary according to the type of administration

(oral administration, parental administration, intravenous or tradermal administration, etc.) .

[0038] Therefore, another aspect of the present invention is related to a pharmaceutical composition comprising said immunogen, an epitope of said immunogen (possibly combined with a carrier molecule such as BSA) and a pharmaceutically adequate carrier suitable for an administration. [0039] Said pharmaceutical composition may comprise also one or more adjuvants which will modulate the immune response of the patient .

[0040] Adjuvants can be any form suitable for administration m human beings . Examples of adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate, such as alum, incomplete Freund's adjuvant, heparm, lyposm, saponm, squalene. Recent reviews on adjuvants for human administration are descried by Gupta R.K. et al . , [ Vaccine 11, pp 293-306 (1993)) and by Johnson A.G. { Clin . Microbiol . Rev. 7, pp 277-289 (1994) ) .

[0041] The pharmaceutical composition according to the invention is prepared by the methods generally applied by the man skilled m the art, for the preparation of various pharmaceutical compositions, especially vaccines, wherein the" percentage of the active compound/pharmaceutical acceptable carrier can vary within very large ranges (generally a suitable dosage unit form contains about 0.005 mg to about 1 mg of compound per kg/body weight of patient) , only limited by the tolerance of the patient to the compound, m particular by the frequency of administration, and by the specific allergies and the symptoms to be treated. [0042] Preferably, the active compound comprising the immunogen is present the pharmaceutical composition a concentration which allows at least the reduction or the suppression of the signs and symptoms of allergy or of a disease of allergic origin [0043] Said pharmaceutical composition could be also a vaccine suitable for treating or preventing allergy, especially airborne and foodborne allergy, as well as diseases of allergic origin. Therefore, a more specific aspect concerns said pharmaceutical composition for use as a medicament, especially for treating and/or preventing allergy, airborne and foodborne allergies as well as diseases of allergic origin. [0044] The present invention is also related to a method comprising the step of administrating to a mammal patient, including a human, a sufficient amount of said pharmaceutical composition for reducing or suppressing the symptoms of allergy.

[0045] A last aspect of the present invention is related to the use of said pharmaceutical composition for the manufacture of a medicament m the prevention and/or the treatment of allergy, especially airborne and foodborne allergies and diseases of allergic origin. [0046] The present invention will be described m detail m the following example, presented as preferred an non-limitmg illustration of the invention.

Example 1 of the invention

[0047] Der p II is a mam, ubiquitous protein which belongs to Group 2 allergens derived from house dust mites (see for instance Bush RK. , Molecular biology of allergens, in Molecular biology of allergy and immunology, Immunology and Allergy Clinics of North America, vol 16, 3, pp 535- 563, 1996) . The latter are involved m allergic sensitization of more than 70% of subjects suffering from respiratory allergy, i.e. rhmo-smusitis and/or bronchial asthma .

[0048] In the course of studies on the immunogenicity of this allergen, the Inventors have identified a major T cell epitope on Der p II, which is recognized by virtually all individuals withm a animal species The sequence of this epitope is SEPCIIHRGKP and will be called hereinafter "SEQ ID NO. 1". [0049] A search for sequence homology identified another universal T cell epitope, called hereinafter "FIS" or "SEQ ID NO. 2", derived from sperm whale myoglobm, the sequence of which is FISEAIIHVLHSR (homologous residues are underlined) . SEQ ID NO. 1 and SEQ ID NO. 2 are phylogenetically unrelated. Myoglobm is the red pigment which constitutes the oxygen reservoir of muscle cells and as such is a major constituent of meat.

[0050] In order to determine experimentally whether this sequence homology could result m functional cross- reactivity between Der p II and myoglobm, mice were immunized by subcutaneous injections of FIS (SEQ ID NO. 2) or myoglobm.

[0051] The FIS (or SEQ ID NO. 2) peptide was obtained by synthesis. Sheep myoglobm was purchased from Sigma Chemicals (St-Louis, MI) .

[0052] Balb/c mice (H-2 ) were injected once m the footpad with 55 μl of a solution containing 100 μg of peptide or myoglobm emulsified m complete Freund' s adjuvant. Ten days later, regional lymph nodes were retrieved, teased and extensively washed with sterile saline to recover cells. The cell suspension was then washed RPMI buffer (Gibco) . Pure CD4⁺ T cell suspensions were obtained by negative selection using antibodies towards CD45R, CDllb and CDllc, all conjugated to magnetic beads according to the manufacturer's instructions (Miltenyi Biotech GmbH, Germany) .

[0053] Functional cross-reactivity between Der p II, FIS and myoglobm was checked by m vi tro proliferation assays Thus, the T cell suspension was adjusted to 5x10 cells per ml m RPMI 1640 buffer supplemented with 2 mM glutam e, 100 U penicillm/ml , 100 μg streptomycm/ml , 5% heat-mactivated fetal calf serum and 5x10 M 2- mercaptoethanol One hundred μl of the T-cell -enriched fraction (4x10 cells per well) was cultured m 100 μl of the above-described medium containing different concentrations of test peptide or myoglobm, and mitomycm- C treated syngeneic naive spleen cells as a source of APC (4x10 per well) . Incubation was carried out into a humidified atmosphere of 5% CO2 m air for 4 days, and cells were pulsed during a final 18-h incubation with 1 μCi of [ H] thymidme per well. The cells were then harvested onto glass fibres using a multiple-cell harvester Radioactivity incorporated into proliferating cells was determined m a liquid scintillation counter. All tests were carried out m triplicate. The proliferative response was expressed as c.p.m.xlO after subtracting background value incorporation m the absence of antigen. [0054] The results showed that T cells from FIS- and myoglobm- immunized mice proliferated to myoglobm and to

Der p II. It has been shown that the FIS peptide, as well as myoglobm, are immunogen.

[0055] As FIS is an epitope of sperm whale myoglobm, the Inventors have checked whether other myoglobm molecules contain close or identical sequences. [0056] A high homology was found for bovine (81%), sheep (82%) , goat (80%) and human myoglobm as determined from the consultation of gene databanks (SWISSPROT protein database) .

[0057] The Der p II T cell epitope, SEQ ID NO 1, described here was shown to be recognised the context of three additional mouse H-2 haplotypes other than (H-2 ) haplotype, as well as by a significant proportion of human subjects (more than 85%) .

[0058] For mouse T cells, a procedure identical to that described above was used with mice pertaining to the

H-2^b (C57BL/6 strain) , H-2^k (C3H strain) and H-2^d (SJL strain) . A significant proliferative response was observed for the three haplotypes . [0059] Moreover, T cells were prepared from the peripheral blood of atopic individuals. Thus, a 50 -ml sample of peripheral blood was obtained m heparin. The blood sample was diluted vol/vol m RPMI 1640 and applied to a Ficoll-Hypaque density gradient. After a 30 mm centπfugation, cells from the interface were collected and washed RPMI 1640. The cell pellet was finally resuspended m RPMI supplemented with 10% AB+ human serum, 80 μg/ml gentamycme, 2 mM L-glutamme and 18 mM D(+)- glucose . Proliferation assays was then carried out as described above for mouse cells, except that the various concentrations of FIS peptide or myoglobm were added immediately to the cell suspension.

[0060] Taken altogether, the obtained results indicate that the phenomenons observed m an animal species are relevant to human beings. [0061] These results show that significant structural and functional cross-reactivity can exist between two phylogenetically totally unrelated proteins and that sensitization to one of such proteins, or to a part of such protein containing an immunogen, can modulate the immune response towards the other. Thus, a food antigen (myoglobm) , or a portion of it containing the appropriate immunogen, can modulate sensitization to an airborne allergen (Der p II) .

Claims (10)

1. Method for modulating the immune system of a mammal patient towards an allergen, which comprises the step of inducing a pre-sensitisation of the immune system of said patient towards an immunogen carrying at least one T cell epitope homologous and functionally similar to an epitope present on said allergen and deriving from a naturally-occurring antigen m order to modify the response of said patient to a further sensitisation to said allergen, allowing that the immune response towards the allergen is modulated and that no allergy towards said allergen will be developed m said patient.

2. Pharmaceutical composition comprising an adequate pharmaceutical carrier and an immunogen derived from a naturally occurring antigen present m a food or a pharmaceutical component, said immunogen corresponding to or carrying at least one T-cell epitope homologous and functionally similar to a T cell epitope present on said allergen.

3. Pharmaceutical composition according to the claim 2, characterised m that said T cell epitope, homologous and functionally similar to a T cell epitope present on said allergen, is present upon a carrier molecule such as BSA.

4. Pharmaceutical composition according to claim 2 or 3 , characterised m that said allergen is an airborne allergen or a foodborne allergen.

5. Pharmaceutical composition according to claim 4, characterised m that said allergen is selected from the group consisting of rhinitis allergens, allergic bronchial asthma allergens and atopic dermatitis allergens.

6. Pharmaceutical composition according to any one of the preceding claims 2 to 5, characterised m that said allergen is the Derp II protein having the T cell epitope identified by SEQ ID NO. 1.

7. Pharmaceutical composition according to any one of the preceding claims 2 to 6, wherein the immunogen is derived from Myoglobm having the T cell epitope identified by SEQ ID NO. 2.

8. Pharmaceutical composition according to any one of the preceding claims 2 to 7 for treating and/or preventing allergy and diseases of allergic origin.

9. Method for the treatment and/or the prevention of allergy comprising the step of administrating a sufficient amount of the pharmaceutical composition according to any of the preceding claims 2 to 7 to a mammal patient (including the human) for decreasing or suppressing the symptoms of an allergy or a disease of allergic origin affecting said patient.

10. Use of the pharmaceutical composition according to any one of the preceding claims for the manufacture of a medicament m the treatment and/or the prevention of allergy and diseases of allergic origin.